Oligonucleotides with a bacterial-like “CpG” have been shown to trigger proinflammatory responses through activation of Toll-like receptor 9 (TLR9). This results in activation of a signal transduction pathway involving the Myd88 adaptor protein, and downstream activation of mitogen activated protein kinase (MAPK) family members and the nuclear factor (NF)-kappa B pathway. This stimulation leads to an increased production of cytokines including interferons, interleukin (IL)-6 and 12, tumor necrosis factor-alpha (TNF-α), chemokines, costimulatory molecules, and antigen presentation on antigen presenting cells (APCs). This process further stimulates adaptive immunity and potentiates a T-helper 1 (Th1) type response.
The cellular and humoral immune responses stimulated by exposure to CpG oligonucleotides is likely a result of the relative abundance of unmethylated CpG motifs in bacteria and other pathogens, as compared to mammalian DNA, in which such motifs are rare. The human immune system has apparently evolved to recognize CpG sequences as early signs of infection and to initiate an immediate immune response against pathogens without causing adverse reactions frequently seen with other proinflammatory agents. Thus, CpG containing nucleic acids, relying on this innate immunity, can utilize a distinct and natural pathway for immune therapy.
Oligonucleotides containing CpG motifs have been developed to exploit this process for use for the prevention and treatment of infections (Dalpke et al., BioDrugs. 2002; 16:419-31, incorporated herein by reference); vaccine adjuvants (Rothenfusser et al., Curr Opin Mol. Ther. 2003. 5:98-106, incorporated herein by reference), including those for the induction of mucosal immunity (Curr Opin Investig Drugs. 2004.5:141-5); allergy (Gangloff and Guenounou. Clin Rev Allergy Immunol. 2004. 26:115-25, incorporated herein by reference); cancer (Jaing et al., Curr Opin Mol. Ther. 2003. 5:180-5; Wang et al., Curr Pharm Des. 2005. 11:2889-907, both incorporated herein by reference); HIV (Becker. Virus Genes. 2005. 30:251-66, incorporated herein by reference); and autoimmune diseases such as lupus (Lenert, Clin Exp Immunol. 2005. 140:1-10, incorporated herein by reference). Importantly proinflammatory CpG motifs show optimal activity in both mouse and human systems without the need to change sequences, suggesting an overriding of the species-dependent specificity of the receptor by the use of synthetic motifs (Kandimalla et al., Biochem Soc Trans. 2003. 31:654-8, incorporated herein by reference).
The activity of CpG proinflammatory nucleic acids can be tested in various immune cell assays. Such methods are described in detail in PCT Published Patent Applications PCT/US95/01570 (WO 96/02555) and PCT/US97/19791 (WO 98/18810) claiming priority to U.S. Ser. Nos. 08/386,063 and 08/960,774, filed on Feb. 7, 1995 and Oct. 30, 1997 respectively. The methods require the use of primary cells from either mice or humans. The effectiveness of the assay on determining the activity of a nucleic acid is dependent upon the appropriate receptors and signaling pathways being present in the cells used.
Recently, research has shown that oligonucleotides devoid of CpG motifs are capable of eliciting a proinflammatory response in vivo and in vitro. Using a mouse knockout model, it was demonstrated that the response to these oligonucleotides is mediated by TLR9. It was suggested that the immunostimulatory motif consists of a 5′-TC dinucleotide in a thymidine rich background, preferably about at least 35% (Vollmer et al., Immunol. 2004. 113:212-223, incorporated herein by reference). The motif was found to stimulate B-cell activation, but lacked a Th1-like cytokines and chemokines.
Oligonucleotides intended for antisense applications are designed to avoid CpG and other proinflammatory motifs that induce a proinflammatory response by using various chemical modifications (i.e. 2′MOE sugars and methylation of cytosine residues) (Henry et al., J. Pharmacol. Expt. Thera. 2000. 292: 468-479). However, some oligonucleotides have immunostimulatory activity, despite having no identifiable proinflammatory motifs and/or containing modified nucleobases to reduce immunostimulatory activity. Such activity is often not identified until the oligonucleotide is administered to an animal. An in vitro method for identifying such oligonucleotides would be desirable.